Luminal Oxidative Regulation of the Ryanodine Receptor: More Sides to the Story?

Ryanodine receptors (RyR) are integral membrane proteins, localized primarily to the membrane of the junctional sarcoplasmic reticulum (jSR). Their topology is asymmetrical in relation to the membrane plane, with approximately 80% of the protein mass facing the cytoplasmic side and an estimated 3% facing the SR lumen.¹ Four RyR molecules oligomerize to form a tetrameric channel that provides the main pathway for the calcium (Ca²⁺) stored in the SR to exit and reach the contractile machinery, a key step in excitation-contraction coupling. RyR2 is the cardiac isoform, and its channel activity is tightly regulated by numerous interacting proteins and post-translational modifications (PTMs) including oxidation, phosphorylation and S-nitrosylation.² While multiple residues have been identified as substrates for PTM, many others are likely.^3,4 Of note, most active residues characterized so far are localized on the cytoplasmic side of the channel, while less is known regarding PTMs and channel regulation via the luminal side.

Proper intracellular Ca²⁺ homeostasis is critical to cardiac function, and disruption in Ca²⁺ equilibrium can lead to life-threatening alterations of the heart rhythm. As a major gatekeeper of intracellular Ca²⁺ storage, RyR2 plays a central role in maintaining electrical synchrony. Mutations in RyR2 or changes in its PTM state can lead to excessive (or reduced) exit of Ca²⁺ from the SR, with secondary consequences such as induction of depolarizing electrogenic currents that can initiate life-threatening arrhythmias.^4,5 Moreover, intracellular Ca²⁺ overload due to leaky RyR2 channels can increase Ca²⁺ uptake into mitochondria, breaking the interplay between Ca²⁺ and oxidative stress.⁴ In particular, mitochondrial Ca²⁺ overload promotes reactive oxygen species (ROS) generation. ROS, specifically H₂O₂, can reach the SR and oxidize RyR2, enhancing its eagerness to leak Ca²⁺ and facilitating a negative feedback loop.⁶

Excessive mitochondrial ROS is a main contributor to ROS-induced disruption of intracellular Ca²⁺ homeostasis. In this issue of Circ Res, Hamilton et al report a study in which they take the innovative approach of examining “the other side” of the RyR2 channel, namely, the SR lumen.⁷ Using a rat model of hypertrophy (thoracic aorta banding; TAB) the authors examine whether members of the oxidoreductase family of proteins that dynamically regulate the oxidative environment within the SR can modulate RyR2. Their data lead them to focus on the luminal oxidoreductase Ero1α, which is induced during ER stress and whose upregulation increases H₂O₂ production within the ER. Through this systematic and careful study, the authors demonstrate a novel regulatory axis for RyR2 ROS modulation involving the luminal ROS sensor Ero1α and the luminal RyR2-binding protein ERp44. This is an impactful study, as it brings to the fore the importance of the SR as an additional ROS source and an important modulator of a channel critical for cardiac electrical homeostasis.

The methodological approach of Hamilton et al is elegant and robust. The authors found that expression levels of the SR oxidoreductase Ero1α were elevated in TAB hearts To study oxidation levels on the luminal side of the SR, they used an SR-targeted, genetically-encoded ROS biosensor (ERroGFP), introduced into Sham and TAB ventricular myocytes (VMs) via adenovirus infection. The data showed an increase in SR oxidation in TAB VMs, which could be reduced by preincubation of the TAB VMs with a specific Ero1α inhibitor (EN460). These molecular observations were complemented with ex vivo optical mapping experiments, which showed that ventricular fibrillation (VF) occurred in all TAB hearts treated with Isoproterenol, but pretreatment with EN460 markedly reduced VF induction. Considering the critical role that the autonomic nervous system can have in the genesis of cardiac arrhythmias, it would be very interesting to perform these sets of experiments also in an in vivo setting, further adding translational value to the study.

To explore the underlying arrhythmia mechanisms in more detail, Hamilton et al. studied Ca²⁺ transients in isolated rat VMs.⁷ Cells from TAB hearts showed reduced Ca²⁺ transient amplitude, an effect reversed by inhibition of Ero1α with EN460. In addition, VMs from hearts pretreated with EN460 did not show burst-induced Ca²⁺ delayed aftertransients, thus suggesting that the antiarrhythmic effect of EN460 stems from suppression of spontaneous Ca²⁺ release events. Separate experiments showed that oxidation levels within the SR were increased in Sham VMs by overexpression of Ero1α, whereas Ero1α knockdown in TAB VMs reduced the redox status to Sham levels and reduced pro-arrhythmic RyR2-mediated spontaneous SR Ca²⁺ release. Measurements of spontaneous Ca²⁺ release were mainly based Ca²⁺ transients and SR load. Future studies may consider analyzing Ca²⁺ sparks under these experimental conditions, to further validate the importance of RyR2 regulation in the process. In addition, it would be interesting to test the antiarrhythmic effect of Ero1α inhibition in murine models developed to study catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular cardiomyopathy (ARVC), two inheritable disorders in which RyR2 function and regulation have been proven critical to the genesis of life-threatening arrhythmic events.^4,8

In their study, Hamilton and colleagues present reduced signal overlap of RyR2 and ERp44 in TAB VMs, which could be increased by EN460, suggesting that the association of ERp44 with RyR2 is redox- and Ero1α-sensitive.⁷ From these and other results they reasoned that interaction of specific RyR2 Cysteine residues with ERp44 underlies luminal Ca²⁺ regulation of the channel, and that this interaction is disrupted by Ero1α overexpression, a hypothesis validated by assessment of an RyR2 Cys4806Ser substitution in HEK293 cells. In silico docking studies further showed that RyR2-Cys4806 and ERp44-Cys29 can associate to form a disulfide bridge. These data suggest that RyR2-Cys4806 is an important mediator of interaction with ERp44, possibly forming a disulfide bridge with Cys29 of ERp44 at normal luminal redox states. This model strongly supports the novel hypotheses that a) binding of ERp44 at RyR2-Cys4806 is necessary to proper RyR2 function, and b) dissociation of ERp44 from RyR2 could lead to arrhythmogenic RyR2 Ca²⁺ release.

Hyperactivity of RyR2 due to posttranslational oxidative modifications plays a key role in Ca^2+- dependent arrhythmogenesis in multiple cardiac diseases. Reducing sudden cardiac death due to ventricular tachyarrhythmias remains a major challenge. Studies using antioxidants or ROS scavengers on isolated cardiomyocytes routinely demonstrate partial improvements of Ca²⁺ homeostasis.⁹ Yet, these findings have not translated into effective treatment strategies, and clinical trials aimed to improve intracellular redox balance have failed to attenuate Ca²⁺-dependent arrhythmogenesis. In this study, Hamilton and colleagues show that antioxidant treatment to reduce RyR2 reactive cysteines at the cytosolic face of the channel only partially recovers RyR2 function, which might explain the limited success of clinical trials.^7,10 Ero1α inhibition significantly reduced malignant ventricular tachyarrhythmias induced by β-adrenergic stimulation in ex vivo hypertrophic rat hearts, suggesting that Ero1α may be a promising therapeutic target to reduce arrhythmogenesis and improve cardiac function. Importantly, Ero1α knockdown in TABs reduced mitochondrial matrix H₂O₂ levels, which suggests that stabilizing SR Ca²⁺ leak can decrease mitochondrial Ca²⁺ overload and prevent ROS generation. However, it is worth remembering that numerous cardiomyopathies present per se mitochondrial dysfunction with the concomitant increased mitochondrial ROS. Mitochondrial ROS is still the major source of oxidative stress in the cell. Thus, the question arises as to whether Ero1α inhibition in vivo could by itself attenuate both SR Ca²⁺ leak and mitochondrial dysfunction. On the other hand, combination treatment with ROS scavengers plus Ero1 inhibitors may be an approach deserving further consideration. Based on the data presented in this interesting article, in vivo studies to evaluate Ero1α as a promising therapeutic target seem well justified. Moreover, exploring the effectiveness of these treatments under conditions of exercise training or adrenergic stress may be particularly interesting, given the importance of cell metabolism and Ca²⁺ regulation on catecholaminergic arrhythmias such as CPVT and ARVC.^8,11

We congratulate the authors on their interesting study. It adds to a body of evidence indicating that post-translational oxidative modifications of RyR2 are as important trigger for catecholaminergic-related arrhythmias in the heart. Moreover, it adds the novel and impactful concept that in addition to well established sources such as NOX2 or mitochondria, the SR is a significant source of ROS that can regulate RyR2 function. The finding of a novel axis of intraluminal interaction between RyR2, ERp44 and Ero1α may open the door for new therapeutic approaches.

Figure 1; Graphic illustration of the positive feedback loop via which mitochondrial dysfunction causes intracellular calcium handling disturbances.

Oxidative stress induces mitochondrial calcium (Ca²⁺) overload via the MCU. Mitochondrial Ca²⁺ overload increases reactive oxygen species (ROS) production and oxidizes the ryanodine receptors (RyR2), causing sarcoplasmic reticulum (SR) Ca²⁺ leakage. Oxidative stress in the SR increases Ero1α expression, causing dissociation of ERp44 from RyR2 and separately stimulating RyR2 Ca²⁺ leakage. Excessive SR Ca²⁺ release creates elevated cytosolic Ca²⁺ levels and free Ca²⁺ ions will be transported back into the mitochondria via the mitochondrial Ca²⁺ uniporter (MCU). Disturbed intracellular Ca²⁺ dynamics will eventually lead to triggered activity and cardiac arrhythmias. Ero1α knockdown or inhibition by EN460 can reduce oxidation levels, restore the RyR2-ERp44 interaction and reduce SR Ca²⁺ leakage. LTCC, l-type calcium channel; RyR2, ryanodine receptor; SERCA, sarco/endoplasmic reticulum Ca²⁺-ATPase; PLN, phospholamban; MCU, mitochondrial Ca2+ uniporter; ROS, reactive oxygen species.